Thermostable or thermoactive DNA polymerase molecules with attenuated 3′-5′ exonuclease activity

Abstract

The present invention provides thermostable or thermoactive DNA polymerases with attenuated 3′-5′ exonuclease activity, methods for their synthesis, methods for their use, kits comprising the polymerases, nucleic acids encoding the polymerases and cells comprising such a nucleic acid. The DNA polymerases of the invention are useful in many recombinant DNA techniques, such as nucleic acid amplification by the polymerase chain reaction. The DNA polymerases of the invention allow higher fidelity replication and amplification of a template DNA sequence, allow less degradation of primers and/or more efficient use of deoxynucleotide triphosphates and are in general more efficient and less costly to make and use.

1. An isolated thermostable or thermoactive DNA polymerase comprising a 3′-5′ exonuclease domain and exhibiting an attenuated 3′-5′ exonuclease activity of about 6.5 or less, but greater than 0, U/pmol, wherein one unit (U) of 3′-5′ exonuclease activity catalyzes the conversion of 50 pmol of a labeled single-stranded oligonucleotide of SEQ ID NO:80 to shorter length oligonucleotides in 15 minutes at 63° C., the enzymatic activity being measured in a reaction mixture containing 4 pmol (100 nM) of the oligonucleotide of SEQ ID NO: 80 in 50 mM Tricine, pH 8.3, 25 mM KOAc, 5% w/v DMSO, 0.5 mM Mn(OAc) 2  and the polymerase, and incubated at 63° C. for 15 minutes, wherein degradation of the oligonucleotide of SEQ ID NO: 80 is measured, wherein said 3′-5′ exonuclease domain comprises a sequence motif selected from the following sequence motifs:
 (a) A sequence motif having the formula DX 1 EX 2 X 3 SX 4 , wherein
 D is an aspartate residue, 
 X 1  is any amino acid residue 
 E is a glutamate residue, 
 X 2  is a threonine, 
 X 3  is any amino acid residue, 
 S is a serine residue, and 
 X 4  is a leucine or an alanine, or 
 
 (b) A sequence motif having the formula X 5 X 6 X 7 X 8 X 9 X 10 X 11 X 12 X 13 X 14  (SEQ ID NO:1), wherein
 X 5  is a glutamine or an alanine, 
 X 6  is an asparagine or an alanine, 
 X 7  is a cysteine or a leucine residue, 
 X 8  is a lysine, 
 X 9  is a phenylalanine or a tyrosine residue, 
 X 10  is an aspartate or a glutamate, 
 X 11  is any amino acid residue, 
 X 12  is any amino acid residue, 
 X 13  is an isoleucine or valine residue, and 
 X 14  is a leucine or phenylalanine residue. 
 
 
     
     
       2. An isolated thermostable or thermoactive DNA polymerase comprising a 3′-5′ exonuclease domain and having a 5′-3′ polymerase activity and an attenuated 3′-5′ exonuclease activity wherein the ratio of said 5′-3′ polymerase activity in U/pmol to said 3′-5′ exonuclease activity in U/pmol is between about 100 and 1, wherein one unit (U) of 3′-5′ exonuclease activity catalyzes the conversion of 50 pmol of a labeled single-stranded oligonucleotide of SEQ ID NO:80 to shorter length oligonucleotides in 15 minutes at 63° C. wherein the 3′-5′ exonuclease activity is measured in a reaction mixture containing 4 pmol (100 nM) of the oligonucleotide of SEQ ID NO: 80 in 50 mM Tricine, pH 8.3, 25 mM KOAc, 5% w/v DMSO, 0.5 mM Mn(OAc) 2  and the polymerase, and incubated at 63° C. for 15 minutes, wherein degradation of the oligonucleotide of SEQ ID NO: 80 is measured, and one unit of 5′-3′ polymerase activity is the amount of enzyme activity required to incorporate 10 nmoles of dNMP into TCA-precipitable DNA product in 30 minutes at 74° C. wherein the 5′-3′ polymerase activity is measured in a reaction mixture containing 25 mM TAPS, pH 9.4, 50 mM KCl, 2 mM MgCl 2 , 1 mM β-mercaptoethanol, 200 μM d(GTA)TP, 100 μM α- 33 P-dCTP, 30 μg activated salmon sperm DNA and the polymerase, and incubated at 74° C. for 10 minutes wherein the amount of TCA precipitable DNA is measured, set forth in Example 3 for 5′-3′ polymerase activity, and said 3′-5′ exonuclease domain comprises a sequence motif selected from the following sequence motifs:
 (a) A sequence motif having the formula DX 1 EX 2 X 3 SX 4 , wherein
 D is an aspartate residue, 
 X 1  is any amino acid residue, 
 E is a glutamate residue, 
 X 2  is a threonine, 
 X 3  is any amino acid residue, 
 S is a serine residue, and 
 X 4  is a leucine or an alanine, or 
 
 (b) A sequence motif having the formula X 5 X 6 X 7 X 8 X 9 X 10 X 11 X 12 X 13 X 14  (SEQ ID NO:1), wherein
 X 5  is a glutamine or an alanine, 
 X 6  is an asparagine or an alanine, 
 X 7  is a cysteine or a leucine residue, 
 X 8  is a lysine, 
 X 9  is a phenylalanine or a tyrosine residue, 
 X 10  is an aspartate or a glutamate, 
 X 11  is any amino acid residue, 
 X 12  is any amino acid residue, 
 X 13  is an isoleucine or valine residue, and 
 X 14  is a leucine or phenylalanine residue. 
 
 
     
     
       3. An isolated modified thermostable or thermoactive DNA polymerase wherein said modified polymerase comprises a 3′-5′ exonuclease domain, has about 0.1 to 65% of a 3′-5′ exonuclease activity of the DNA polymerase before modification, wherein one unit (U) of 3′-5′ exonuclease activity catalyzes the conversion of 50 pmol of a labeled single-stranded oligonucleotide of SEQ ID NO:80 to shorter length oligonucleotides in 15 minutes at 63° C., the enzymatic activity being measured in a reaction mixture containing 4 pmol (100 nM) of the oligonucleotide of SEQ ID NO: 80 in 50 mM Tricine, pH 8.3, 25 mM KOAc, 5% w/v DMSO, 0.5 mM Mn(OAc) 2  and polymerase, and incubated at 63° C. for 15 minutes, wherein degradation of the oligonucleotide of SEQ ID NO. 80 is measured, and said 3′-5′ exonuclease domain comprises a sequence motif selected from the following sequence motifs:
 (a) A sequence motif having the formula DX 1 EX 2 X 3 SX 4 , wherein
 D is an aspartate residue, 
 X 1  is any amino acid residue 
 E is a glutamate residue, 
 X 2  is a threonine, 
 X 3  is any amino acid residue, 
 S is a serine residue, and 
 X 4  is a leucine or an alanine, or 
 
 (b) A sequence motif having the formula X 5 X 6 X 7 X 8 X 9 X 10 X 11 X 12 X 13 X 14  (SEQ ID NO:1), wherein
 X 5  is a glutamine or an alanine, 
 X 6  is an asparagine or an alanine, 
 X 7  is a cysteine or a leucine residue, 
 X 8  is a lysine, 
 X 9  is a phenylalanine or a tyrosine residue, 
 X 10 is an aspartate or a glutamate, 
 X 11  is any amino acid residue, 
 X 12  is any amino acid residue, 
 X 13  is an isoleucine or valine residue, and 
 X 14  is a leucine or phenylalanine residue.